Bioburden Method Suitability for Cleaning and Sanitation Monitoring: How Far Do We Have to Go? - Pharmaceutical Technology

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Bioburden Method Suitability for Cleaning and Sanitation Monitoring: How Far Do We Have to Go?
The author reviews test methods for microbiological cleaning processes and suggests ways to improve microbial bioburden method suitability studies.

Pharmaceutical Technology
Volume 34, Issue 8

The test method suitability perspective

The validation of surface-recovery methods (i.e., chemical and microbiological) is a pre-requisite for residual determination of cleaning effectiveness in process validation studies. These methods should be challenged in the laboratory using pilot-scale controlled conditions in order to evaluate the suitability for their intended use. For this purpose, validation specialists select representative surfaces identified within the production area and potentially in contact with ingredients, product intermediates, and bulk products. Surfaces selected for method validation commonly include stainless steel 316L, glass, plastic (such as polyvinyl-chloride and polyethylene), and some metal alloys. However, surface selections for challenging studies are not justified based on what really matters: demonstrating the effectiveness of the monitoring method (16, 17).

Differences in surfaces

There are many types of surfaces in the pharmaceutical production areas and current-good-manufacturing-practice (CGMP) equipment, all with distinct physico-chemical properties. Most of these surfaces are well defined. When microorganisms are released into the manufacturing area, they will be deposited onto these surfaces as either aerosol particles or as liquid droplets. The type of surface greatly influences their ability to survive and their possibility to contaminate other materials.

Surfaces can be split into several groups, for example, porous and nonporous, inert or active, rough or smooth, and hydrophobic or hydrophilic. Glass and stainless steel are examples of nonporous inert surfaces, and galvanized steel, brass, and copper are example of nonporous active surfaces (18, 19). Stainless steel has been extensively studied, in part because it is the principal material of construction of good manufacturing practice (GMP) equipment (29). Microscopically, stainless steel may show grooves and crevices that can trap bacteria but glass does not (20). Some bacteria have been found to be able to adhere to stainless-steel surfaces after short contact times if the conditions are appropriate (i.e., adequate temperature and humidity) (21).

The porosity of a surface is a major factor affecting bacteria adherence. Highly porous surfaces facilitate adherence of bacteria. However, the adherence of bacteria depends on the number of cells—the higher the number of cells, the higher the probability those cells remain attached on surface after rinse (22). It was demonstrated that gram-negative bacteria adhesion could be decreased with the addition of silicone on porous material such as plastics, Teflon, and Dacron (19). Additionally, it was reported that rubber and plastic coupons were significantly more accessible to bacteria than glass coupons, as revealed by the high population of bacteria recovered from their surfaces (23). Porous materials such as plastics, Teflon, Dacron, and their combination are used less often as materials of construction in GMP equipment. Rijnaarts and colleagues reported that bacteria deposition on Teflon is faster than on glass.

Silicone rubber has found widespread use in medical, aerospace, electrical, construction, and industrial applications (24). Silicone rubbers are synthetic polymers with a giant backbone of alternating silicon and oxygen atoms. The nonporous nature of silicone's surface does not allow the adhesion of bacteria (25, 26). Nevertheless, studies of bacterial adhesion with laboratory strains of bacteria (i.e., type culture collection strains), many of which had been transferred thousands of times and lost their ability to adhere, first indicated that very smooth surfaces might escape bacterial colonization. Subsequent studies with "wild" and fully adherent bacterial strains showed that smooth surfaces are colonized as easily as rough surfaces and that the physical characteristics of a surface influence bacterial adhesion to only a minor extent (27). This point is important to remember when selecting test microorganisms for suitability testing.

Porosity may prevent water evaporation. The lethal effect of desiccation was found to be the most important death mechanism in bacteria (26). Similar studies performed on a Teflon surface using Escherichia coli, Acinetobacter sp., Pseudomonas oleovorans, and Staphylococcus aureus demonstrated that all four species survived well during the droplet evaporation process, but died mostly at the time when droplets were dried out at 40 to 45 min (26). However, some non-spore-forming bacteria might be able to withstand dry conditions on surfaces for an extended period of time (15). Kusumaningrum and colleagues concluded that the survival of microbes under these conditions is dependent on the contamination levels and type of pathogen. High-density polyethylene (HDPE) is prepared from ethylene by a catalytic process (15). The absence of branching results in a more closely packed structure with a higher density and somewhat higher chemical resistance than low-density polyethylene (LDPE). HDPE is chemically inert and the surface is more porous than LDPE. HDPE is used in products and packaging detergent bottles, garbage containers and water pipes (28).

Fabrics are porous surfaces (e.g., cotton, polyester, polyethylene, polyurethane and their combinations, etc.) that demonstrate survival of gram-negative and gram-positive microorganisms even longer than plastics (29, 30). It has also been observed that gram-positive bacteria survive slightly longer than gram-negatives. Because of their porous nature, it is recommended to rinse fabrics and other porous surfaces in order to detach microbes from them. Swab and plate contact methods are not suitable for fabrics.

It is well known that charged molecules in a solution are able to kill bacteria (31–33). However, it has been realized more recently that charges attached to surfaces can kill bacteria upon contact (34). Certain surfaces such as brass, copper and galvanized steel can be toxic to bacteria because the presence of water and air allows the release of metal ions from metal surface (19), and these metal ions exert an antimicrobial effect by interfering with biological pathways and enzymes (18, 33). Copper releases Cu2+ ions, galvanized steel releases Zn2+ ions, and brass releases both Cu2+ and Zn2+ ions. These metal ions are, in fact, essential micronutrients of bacterial cells at very low concentrations. Above these levels they may exert a toxic effect on aerobic bacteria.

Different types of plastics have different surface properties, which influence their effect on bacteria. Polyvinyl chloride (PVC) and polypropylene (PP) are two similar plastics, but have different properties. PP is more stable and less reactive than PVC. PVC surfaces show high mortality rates for bacteria while PP surfaces show no significant levels of mortality. Studies with Enterococcus faecalis aerosol on PVC and PP demonstrated that PVC had a significant effect on the survival of bacteria due to oxidation reactions with the walls of gram-negative bacteria (30).


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